Estrogen receptor (ER) activity determines growth, survival, and differentiation in ER-positive breast cancer cells through transcriptional control of numerous genes. Although ER is an important therapeutic target, only 20% - 50% of women with metastatic breast cancer that is ER-positive by immunohistochemistry (IHC) will respond to ER-targeted endocrine therapies. We aim to develop a more accurate test than IHC to predict endocrine sensitivity in women with metastatic breast cancer that has recurred after a prior adjuvant or palliative endocrine therapy. We are calling this second-line endocrine therapy. Our hypothesis is that breast cancers with greatest expression and activity of ER are addicted to ER-dependent pathways and are most susceptible to an endocrine therapy, and so an assay that measures the output from ER-related transcription will predict response to endocrine therapy. To address this, we defined a multigene ER reporter index (Rl) from an independent public microarray dataset. Levels of ER mRNA (ESR1) from microarrays accurately diagnosed ER IHC status in FNAs or tissues. Measurements of ESR1 and Rl were reproducible. ESR1 and Rl were both independently related to distant relapse-free survival (DRFS) in women with ER-positive breast cancer who received adjuvant tamoxifen therapy. This association with DRFS was not seen in untreated, node-negative, ER-positive breast cancers, and so cannot be attributed to prognosis. We observed dissociation of the relationship between ER and its transcriptional output (Rl) in ER-positive breast cancers of advanced stage. ESR1 expression levels were similar, but Rl was lower with advancing stage. This may contribute to decreased sensitivity to endocrine therapy in ER-positive metastatic breast cancers. We propose to measure ESR1 and Rl expression values independently, and as a combined reporter index (CRI), in metastatic (stage IV) ER-positive breast cancer and compare those measurements with response to second-line endocrine therapy in general, and also in subsets of patients treated with an aromatase inhibitor or an estrogen receptor modulator. In the future, a more predictive test for endocrine sensitivity in relapsed ER-positive breast cancer would help oncologists to determine when to continue with a sequence of different endocrine therapies for metastatic breast cancer, and when to switch treatment to cytotoxic chemotherapy and/or other molecular therapies. [unreadable] [unreadable] [unreadable]